Is There Something Fishy About Teaching Evolution? Explore Biochemical Evidence for Evolution

2. Is There Something Fishy About Teaching Evolution?Explore BiochemicalEvidence for Evolution

3. Protein Fingerprinting Kit

4. Protein Fingerprinting KitInstructors Stan Hitomi Director, Edward Teller Education Center UC Davis / Lawrence Livermore National Laboratory, Livermore, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Sherri Andrews, Ph.D. North Carolina School of the Arts Winston-Salem, NC

5. WhyTeach Protein Electrophoresis? • Powerful teaching tool • Real-world connections • Laboratory extensions • Tangible results • Link to careers and industry

6. Hands-on Evolution Wet Lab • Analyze protein profiles from a variety of fish • Use acrylamide electrophoresis to separate proteins by size • Compare biochemical and phylogenetic relationships

7. Traditional Systematics and Taxonomy • Classification • Kingdom • Phylum • Class • Order • Family • Genus • Species • Traditional classification based upon traits: • Morphological • Behavioral

8. Can biomolecular evidence be used to determine evolutionary relationships?

9. Biochemical Similarities • Traits are the result of: • Structure • Function • Proteins determine structure and function • DNA codes for proteins that confer traits

10. Biochemical Differences • Changes in DNA leads to proteins with: • Different functions • Novel traits • Positive, negative or no effects • Genetic diversity provides pool for natural selection = evolution

11. Protein Fingerprinting Procedures Day 2 Day 1 Day 3

12. What’s in theSample Buffer? • Tris buffer to provide appropriate pH • SDS (Sodium Dodecyl Sulfate) detergent to dissolve proteins and give them a negative charge • Glycerol to make samples sink into wells • Bromophenol Blue dye to visualize samples

13. Why Heat the Samples? • Heating the samples denatures protein complexes, allowing the separation of individual proteins by size SDS, heat s-s Proteins with SDS – +

14. Making Proteins

15. 1o 2o 3o 4o Levels of Protein Organization

16. Protein Size Comparison • Break protein complexes into individual proteins • Denature proteins using detergent and heat • Separate proteins based on size

17. Protein Size • Size measured in kilodaltons (kDa) • Dalton = mass of hydrogen molecule = 1.66 x 10-24 gram • Average amino acid = 110 daltons

18. Muscle Contains Proteins of Many Sizes

19. Actin and Myosin • Actin • 5% of total protein • 20% of vertebrate muscle mass • 375 amino acids = 42 kDa • Forms filaments • Myosin • Tetramer • two heavy subunits (220 kDa) • two light subunits (20 kDa) • Breaks down ATP for muscle contraction

20. How Does an SDS-PAGE Gel Work? • Negatively charged proteins move to positive electrode • Smaller proteins move faster • Proteins separate by size SDS, heat s-s Proteins with SDS – +

21. CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 O S O O SDS - O SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) • SDS Detergent (Sodium Dodecyl Sulfate) • Solubilizes and denatures proteins • Adds negative charge to proteins • Heat denatures proteins

22. Why Use Acrylamide Gels to Separate Proteins? • Acrylamide gel give a tight matrix • Ideal for protein separation • Smaller pore size than agarose • Proteins much smaller than DNA • Average amino acid = 110 Da • Average nucleotide pair = 649 Da • 1 kilobase of DNA = 650 kDa • 1 kilobase of DNA encodes 333 amino acids = 36 kDa

23. Gel Analysis Lane • Kaleidoscope Markers • Shark • Salmon • Trout • Catfish • Sturgeon • Actin and Myosin Standard

24. kDa mm 203 8.5 135 12.0 86 18.5 41 28.0 33 34.0 19 41.5 8 44.5 Molecular Weight Estimation 250 200 150 Size in kDa 100 50 0 0 20 40 60 Distance (mm from well)

25. Molecular Weight Analysis

26. Phylogenetic Tree

27. Extensions • Independent Study • Western blot analysis

28. Ready Gel Assembly Step 1 Step 2 Step 3 Step 4